Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen-Brief Report.

BACKGROUND: Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and ß-sheet regions among species; however, their functions have not been studied. METHODS: Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), ß-sheet (AAV.ßsheet-Mut), or both regions (AAV.loop/ßsheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT-/-) mice in an LDL (low-density lipoprotein) receptor-deficient background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells. RESULTS: In hepAGT-/- mice infected with AAV.loop-Mut or ßsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/- mice infected with AAV.loop/ßsheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/ßsheet-Mut or HepG2 cells transducted with AAV.loop/ßsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. CONCLUSIONS: The conserved sequences in either the loop or ß-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.

Two Amino Acids Proximate to the Renin Cleavage Site of Human Angiotensinogen Do Not Affect Blood Pressure and Atherosclerosis in Mice-Brief Report.

OBJECTIVE: Renin cleavage of angiotensinogen has species specificity. As the residues at positions 11 and 12 are different between human angiotensinogen and mouse angiotensinogen, we determined whether these 2 residues in angiotensinogen affect renin cleavage and angiotensin II-mediated blood pressure regulation and atherosclerosis using an adenoassociated viral approach for manipulating angiotensinogen in vivo. Approach and Results: Hepatocyte-specific angiotensinogen deficient (hepAGT-/-) mice in an LDL receptor-deficient background were infected with adenoassociated virals containing a null insert, human angiotensinogen, or mouse angiotensinogen expressing the same residues of the human protein at positions 11 and 12 (mouse angiotensinogen [L11V;Y12I]). Expression of human angiotensinogen in hepAGT-/- mice led to high plasma human angiotensinogen concentrations without changes in plasma endogenous mouse angiotensinogen, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human angiotensinogen not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human angiotensinogen lead to the inability of mouse renin to cleave human angiotensinogen, hepAGT-/- mice were injected with adenoassociated viral vector encoding mouse angiotensinogen (L11V;Y12I). Expression of mouse angiotensinogen (L11V;Y12I) in hepAGT-/- mice resulted in increased plasma mouse angiotensinogen concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT+/+ mice. This mouse angiotensinogen variant increased blood pressure and atherosclerosis in hepAGT-/- mice to the magnitude of hepAGT+/+ mice. CONCLUSIONS: Replacement of L11 and Y12 to V11 and I12, respectively, in mouse angiotensinogen does not affect renin cleavage, blood pressure, and atherosclerosis in LDL receptor-deficient mice.

miR-146a Deficiency Accelerates Hepatic Inflammation Without Influencing Diet-induced Obesity in Mice.

miR-146a, an anti-inflammatory microRNA, is shown to be a negative regulator of adipocyte inflammation. However, the functional contribution of miR-146a in the development of obesity is not defined. In order to determine whether miR-146a influences diet-induced obesity, mice that were either wild type (WT) or miR-146a deficient (KO) were fed with high (60% kcal) fat diet (HFD) for 16 weeks. Deficiency of miR-146a did not influence obesity measured as HFD-induced body weight and fat mass gain, or metabolism of glucose and insulin tolerance. In addition, adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively, were comparable between the two groups of mice. Although, miR-146a deficiency had no influence on HFD-induced hepatic lipid accumulation, interestingly, it significantly increased obesity-induced inflammatory responses in liver tissue. The present study demonstrates that miR-146a deficiency had no influence on the development of HFD-induced obesity and adipose tissue remodeling, whereas it significantly increased hepatic inflammation in obese mice. This result suggests that miR-146a regulates hepatic inflammation during development of obesity.

Calpain Inhibition Attenuates Adipose Tissue Inflammation and Fibrosis in Diet-induced Obese Mice.

Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not completely defined. Calpains are calcium-dependent neutral cysteine proteases that modulate cellular function and have been implicated in various inflammatory diseases. To define whether activated calpains influence diet-induced obesity and adipose tissue macrophage accumulation, mice that were either wild type (WT) or overexpressing calpastatin (CAST Tg), the endogenous inhibitor of calpains were fed with high (60% kcal) fat diet for 16 weeks. CAST overexpression did not influence high fat diet-induced body weight and fat mass gain throughout the study. Calpain inhibition showed a transient improvement in glucose tolerance at 5 weeks of HFD whereas it lost this effect on glucose and insulin tolerance at 16 weeks HFD in obese mice. However, CAST overexpression significantly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively. CAST overexpression significantly attenuated obesity-induced inflammatory responses in adipose tissue. Furthermore, calpain inhibition suppressed macrophage migration to adipose tissue in vitro. The present study demonstrates a pivotal role for calpains in mediating HFD-induced adipose tissue remodeling by influencing multiple functions including apoptosis, fibrosis and inflammation.

Smooth Muscle Cells Derived From Second Heart Field and Cardiac Neural Crest Reside in Spatially Distinct Domains in the Media of the Ascending Aorta-Brief Report.

OBJECTIVE: Smooth muscle cells (SMCs) of the proximal thoracic aorta are embryonically derived from the second heart field (SHF) and cardiac neural crest (CNC). However, distributions of these embryonic origins are not fully defined. The regional distribution of SMCs of different origins is speculated to cause region-specific aortopathies. Therefore, the aim of this study was to determine the distribution of SMCs of SHF and CNC origins in the proximal thoracic aorta. APPROACH AND RESULTS: Mice with repressed LacZ in the ROSA26 locus were bred to those expressing Cre controlled by either the Wnt1 or Mef2c (myocyte-specific enhancer factor 2c) promoter to trace CNC- and SHF-derived SMCs, respectively. Thoracic aortas were harvested, and activity of ß-galactosidase was determined. Aortas from Wnt1-Cre mice had ß-galactosidase-positive areas throughout the region from the proximal ascending aorta to just distal of the subclavian arterial branch. Unexpectedly, ß-galactosidase-positive areas in Mef2c-Cre mice extended from the aortic root throughout the ascending aorta. This distribution occurred independent of sex and aging. Cross and sagittal aortic sections demonstrated that CNC-derived cells populated the inner medial aspect of the anterior region of the ascending aorta and transmurally in the media of the posterior region. Interestingly, outer medial cells throughout anterior and posterior ascending aortas were derived from the SHF. ß-Galactosidase-positive medial cells of both origins colocalized with an SMC marker, α-actin. CONCLUSIONS: Both CNC- and SHF-derived SMCs populate the media throughout the ascending aorta. The outer medial cells of the ascending aorta form a sleeve populated by SHF-derived SMCs.

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

OBJECTIVE: Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. APPROACH AND RESULTS: To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. CONCLUSIONS: The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Angiotensinogen Exerts Effects Independent of Angiotensin II.

OBJECTIVE: This study determined whether angiotensinogen (AGT) has angiotensin II-independent effects using multiple genetic and pharmacological manipulations. APPROACH AND RESULTS: All study mice were in low-density lipoprotein receptor -/- background and fed a saturated fat-enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were >90% lower compared with their wild-type littermates. HypoAGT mice had lower systolic blood pressure, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in low-density lipoprotein receptor -/- mice. In mice with established adiposity, administration of AGT antisense oligonucleotide versus aliskiren led to equivalent reductions of systolic blood pressure and atherosclerosis. AGT antisense oligonucleotide administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat-enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat, and human revealed 4 highly conserved sequences within the des(angiotensin I)AGT domain. des(angiotensin I)AGT, through adeno-associated viral infection in hepatocyte-specific AGT-deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis. CONCLUSIONS: AGT contributes to body weight gain and liver steatosis through functions of the des(angiotensin I)AGT domain, which are independent of angiotensin II production.

Cys18-Cys137 disulfide bond in mouse angiotensinogen does not affect AngII-dependent functions in vivo.

Renin cleavage of angiotensinogen (AGT) releases angiotensin I (AngI) in the initial step of producing all angiotensin peptides. It has been suggested recently that redox regulation of a disulfide bond in AGT involving Cys18-Cys137 may be important to its renin cleavage efficiency in vivo. The purpose of this study was to test this prediction in a mouse model by comparing AngII production and AngII-dependent functions in mice expressing wild-type AGT versus a mutated form of AGT lacking the disulfide bond. Wild-type (hepAGT+/+) and hepatocyte-specific AGT-deficient (hepAGT-/-) littermates were developed in an low-density lipoprotein receptor -/- background. hepAGT+/+ mice were injected intraperitoneally with adeno-associated viral (AAV) vector containing a null insert. hepAGT-/- mice were injected with AAV containing a null insert, wild-type AGT or Cys18Ser and Cys137Ser mutated AGT. Two weeks after AAV injection, mice were fed a Western diet for 12 weeks. Administration of AAV containing either form of AGT led to similar plasma AGT concentrations in hepAGT-/- mice. High plasma renin concentrations in hepAGT-/- mice were suppressed equally by both forms of AGT, which were accompanied by comparable increases of plasma AngII concentrations similar to hepAGT+/+ mice. AAV-driven expression of both forms of AGT led to equivalent increases of systolic blood pressure and augmentation of atherosclerotic lesion size in hepAGT-/- mice. These measurements were comparable to systolic blood pressure and atherosclerotic lesions in hepAGT+/+ mice. These data indicate that the Cys18-Cys137 disulfide bond in AGT is dispensable for AngII production and AngII-dependent functions in mice.

Amlodipine reduces AngII-induced aortic aneurysms and atherosclerosis in hypercholesterolemic mice.

BACKGROUND: The purpose of this study was to determine effects of amlodipine, a dihydropyridine calcium channel blocker, on development of angiotensin II (AngII)-induced vascular pathologies. METHODS AND RESULTS: Male LDL receptor -/- mice were infused with vehicle, amlodipine (5 mg/kg/d), AngII (1,000 ng/kg/min), or AngII + amlodipine for 4 weeks through osmotic pumps (n=10/group). Mice were fed a saturated fat-enriched diet for 1 week prior to pump implantation and during 4 weeks of infusion. Infusion of amlodipine resulted in plasma concentrations of 32 ± 2 ng/ml and 27 ± 2 ng/ml for mice in saline + amlodipine and AngII + amlodipine groups, respectively. This infusion rate of amlodipine did not affect AngII-induced increases in systolic blood pressure. Three of 10 (30%) mice infused with AngII died of aortic rupture, while aortic rupture did not occur in mice co-infused with AngII + amlodipine. Suprarenal aortic width and intimal area of ascending aortas were measured to define aortic aneurysms. In the absence of AngII infusion, amlodipine did not change suprarenal aortic width and ascending aortic area. Infusion of AngII led to profound increases of suprarenal aortic width (saline + vehicle versus AngII + vehicle: 0.86 ± 0.02 versus 1.72 ± 0.26 mm; P=0.0006), whereas co-infusion of AngII and amlodipine diminished abdominal dilation (1.02 ± 0.14 mm; P=0.003). As expected, AngII infusion increased mean intimal area of ascending aortas (saline + vehicle versus AngII + vehicle: 8.5 ± 0.3 versus 12.5 ± 1.1 mm(2); P=0.001), while co-infusion of AngII and amlodipine ablated dilation of the ascending aorta (8.6 ± 0.2 mm(2); P=0.03). Co-administration of amlodipine also significantly attenuated AngII-induced atherosclerosis in the thoracic region as quantified by percent lesion area (AngII + vehicle versus AngII + amlodipine: 5.8 ± 2.1 % versus 0.3 ± 0.1%; P=0.05). CONCLUSIONS: Amlodipine inhibited AngII-induced aortic aneurysms in both the abdominal and ascending regions, and atherosclerosis in hypercholesterolemic mice.

Calpain-2 compensation promotes angiotensin II-induced ascending and abdominal aortic aneurysms in calpain-1 deficient mice.

BACKGROUND AND OBJECTIVE: Recently, we demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. METHODOLOGY/RESULTS: To investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr -/- mice that were either calpain-1 +/+ or -/- were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and -/- mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an actin binding cytoskeletal protein in aorta. CONCLUSION: Calpain-2 compensates for loss of calpain-1, and both calpain isoforms are involved in AngII-induced aortic aneurysm formation in mice.

Calpain inhibition attenuates angiotensin II-induced abdominal aortic aneurysms and atherosclerosis in low-density lipoprotein receptor-deficient mice.

Chronic infusion of angiotensin II (AngII) augments atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. AngII-induced AAAs are associated with medial macrophage accumulation and matrix metalloproteinase (MMP) activation. Inhibition of calpain, a calcium-activated neutral cysteine protease, by overexpression of its endogenous inhibitor, calpastatin, attenuates AngII-induced leukocyte infiltration, perivascular inflammation, and MMP activation in mice. The purpose of this study was to define whether pharmacological inhibition of calpain influences AngII-induced AAAs in hypercholesterolemic mice. Male low-density lipoprotein receptor-/- mice were fed a fat-enriched diet and administered with either vehicle or a calpain-specific inhibitor, BDA-410 (30 mg/kg per day) for 5 weeks. After 1 week of feeding, mice were infused with AngII (1000 ng/kg per minute) for 4 weeks. AngII-infusion profoundly increased aortic calpain protein and activity. BDA-410 administration had no effect on plasma cholesterol concentrations or AngII-increased systolic blood pressure. Calpain inhibition significantly attenuated AngII-induced AAA formation and atherosclerosis development. BDA-410 administration attenuated activation of MMP12, proinflammatory cytokines (IL-6, monocyte chemoattractant protein-1), and macrophage infiltration into the aorta. BDA-410 administration significantly attenuated thioglycolate-elicited macrophage accumulation in the peritoneal cavity. We conclude that calpain inhibition using BDA-410 attenuated AngII-induced AAA formation and atherosclerosis development in low-density lipoprotein receptor-/- mice.

Deficiency of receptor-associated protein attenuates angiotensin II-induced atherosclerosis in hypercholesterolemic mice without influencing abdominal aortic aneurysms.

OBJECTIVE: Receptor-associated protein (RAP) was initially described as a regulator of low density lipoprotein receptor-related protein 1 (LRP1), but is now known to regulate many proteins. Since the direct effects of RAP on vascular pathologies have not been studied, this study determined whether RAP deficiency influenced angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. METHODS AND RESULTS: Male LDL receptor -/- mice that were either RAP +/+ or -/- were infused with AngII (500 ng/kg/min) for 4 weeks while consuming a saturated fat-enriched diet. RAP deficiency had no effects on body weight or AngII-induced increases of systolic blood pressure. Despite increased plasma cholesterol concentrations, RAP deficiency reduced atherosclerotic lesion size in aortic arches, while having no effect on AngII-induced AAAs. RAP deficiency profoundly reduced LRP1 protein abundance in macrophages, but did not change its abundance in aortic smooth muscle cells. Also, RAP deficiency had no effects on mRNA abundance of LRP1 or lipoprotein lipase in macrophages. To determine whether RAP deficiency in leukocytes influenced AngII-induced atherosclerosis, irradiated male LDL receptor -/- mice were repopulated with bone marrow-derived cells from either RAP +/+ or -/- male mice. The chimeric mice were infused with AngII (500 ng/kg/min) for 4 weeks while fed the saturated fat-enriched diet. RAP deficiency in bone marrow-derived cells did not influence either plasma cholesterol concentrations or atherosclerotic lesion size. CONCLUSIONS: Whole body RAP deficiency attenuated atherosclerosis without influencing AAAs in hypercholesterolemic mice infused with AngII. The anti-atherogenic effect was not attributable to RAP deficiency in bone marrow-derived cells.

Prolonged infusion of angiotensin II in apoE(-/-) mice promotes macrophage recruitment with continued expansion of abdominal aortic aneurysm.

Angiotensin II (AngII) infusion initiates abdominal aortic aneurysm (AAA) development due to medial disruption and results in luminal dilation and thrombus formation. The objective of this study was to determine whether AAA progressed during protracted AngII infusion. Male apoE(-/-) mice were infused with AngII using miniosmotic pumps. On day 27, suprarenal aortic luminal diameters were ultrasonically measured to identify mice exhibiting AAAs. Mice were designated to three groups with similar mean luminal dilation. Group 1 mice were sacrificed on day 28. Group 2 and 3 mice were subsequently infused with saline or AngII, respectively, for an additional 56 days. In Group 2, saline infusion-after the initial 28 days of AngII infusion-led to an immediate decrease in systolic blood pressure. Over the subsequent 56 days of saline infusion, there were no aneurysm-related deaths or significant changes in luminal diameter. In contrast, continuous AngII infusion in Group 3 maintained persistently increased systolic blood pressure, with aneurysmal rupture-associated deaths, increased luminal diameters, and tissue remodeling. Aortic aneurysmal segments that expanded during continuous AngII infusion exhibited macrophage accumulation in regions of medial disruption, predominantly on the adventitial aspect. Macrophages immunostained for CD206 more than for iNOS, consistent with an M2 phenotype. In conclusion, prolonged AngII infusion promotes AAA expansion, and is associated with enhanced rupture rates and increased macrophage infiltration.

Endothelial cell-specific deficiency of Ang II type 1a receptors attenuates Ang II-induced ascending aortic aneurysms in LDL receptor-/- mice.

RATIONALE: Human studies and mouse models have provided evidence for angiotensin II (Ang II)-based mechanisms as an underlying cause of aneurysms localized to the ascending aorta. In agreement with this associative evidence, we have published recently that Ang II infusion induces aneurysmal pathology in the ascending aorta. OBJECTIVE: The aim of this study was to define the role of angiotensin II type 1a (AT(1a)) receptors and their cellular location in Ang II-induced ascending aortic aneurysms (AAs). METHODS AND RESULTS: Male LDL receptor(-/-) mice were fed a saturated fat-enriched diet for 1 week before osmotic mini-pump implantation and infused with either saline or Ang II (1000 ng/kg per minute) for 28 days. Intimal surface areas of ascending aortas were measured to quantify ascending AAs. Whole body AT(1a) receptor deficiency ablated Ang II-induced ascending AAs (P<0.001). To determine the role of AT(1a) receptors on leukocytes, LDL receptor(-/-)×AT(1a) receptor(+/+) or AT(1a) receptor(-/-) mice were irradiated and repopulated with bone marrow-derived cells isolated from either AT(1a) receptor(+/+) or AT(1a) receptor(-/-) mice. Deficiency of AT(1a) receptors in bone marrow-derived cells had no effect on Ang II-induced ascending AAs. To determine the role of AT(1a) receptors on vascular wall cells, we developed AT(1a) receptor floxed mice with depletion on either smooth muscle or endothelial cells using Cre driven by either SM22 or Tek, respectively. AT(1a) receptor deletion in smooth muscle cells had no effect on ascending AAs. In contrast, endothelial-specific depletion attenuated this pathology. CONCLUSIONS: Ang II infusion promotes aneurysms in the ascending aorta via stimulation of AT(1a) receptors that are expressed on endothelial cells.

Rapid dilation of the abdominal aorta during infusion of angiotensin II detected by noninvasive high-frequency ultrasonography.

BACKGROUND: Infusion of angiotensin II (AngII) via subcutaneous osmotic pumps into mice promotes the development of abdominal aortic aneurysms (AAAs). These AngII-induced AAAs develop via a complex process in which there is a transmedial break, lumen dilation, thrombus formation, inflammation involving cells of both the innate and acquired immune systems, and remodeling. The recent development of a high-frequency ultrasound machine has permitted the noninvasive detection of murine abdominal aortas. We assessed the ability of a Visualsonics Vevo 660 high-resolution imaging system to detect AAAs and sequentially quantify the aortic luminal diameter. This system had 100% accuracy in detecting AngII-induced AAAs in vivo, with intrauser and interuser variation coefficients of less than 10% for quantification of the aortic lumen diameter. METHODS: Male apolipoprotein E (apoE)(-/-) mice were infused subcutaneously with either saline or AngII and were monitored with this ultrasonic system to define the temporal changes in aortic lumen diameter. Aortic luminal diameters were measured in the aneurysm-susceptible region of the suprarenal aorta. For internal controls, abdominal aortic diameters were measured at the level of the left renal branch, because this landmark region did not dilate during AngII infusion. RESULTS: Luminal diameters of the suprarenal aorta did not change significantly in saline-infused mice over 28 days of measurement (P = .71). In contrast, AngII infusion led to rapid dilation of suprarenal aortas during the initial 7 days of infusion (0.071 mm/d; P = .0037 for the change in the initial expansion rate). Further luminal diameter expansions occurred for the remaining 21 days of observation at a more modest rate (0.023 mm/d; P = .0001 for continued expansion after day 7). Within the initial 14 days of AngII infusion, some apoE(-/-) mice died as a result of rupture of the aorta in the suprarenal region. We had previously assumed that aortic dilation and rupture occurred simultaneously. However, in the AngII-infused mice that succumbed to aortic rupture, luminal diameters increased several days before death. CONCLUSIONS: High-frequency ultrasonography demonstrated that suprarenal aortic expansion occurs rapidly after the initiation of AngII infusion into apoE(-/-) mice.

Angiotensin II infusion induces site-specific intra-laminar hemorrhage in macrophage colony-stimulating factor-deficient mice.

Angiotensin II (AngII) infusion promotes macrophage infiltration into the aortic wall resulting in several forms of vascular pathology. To determine the causal role of macrophages in these vascular diseases, we used osteopetrotic (op) male mice in which a natural mutation ablates production of M-CSF and results in severe depletion of monocytes. AngII infusion into apoE-/- mice resulted in increased atherosclerosis that was attenuated in op mice. AngII infusion in op mice unexpectedly produced grossly discernable thickening of the proximal thoracic aorta characterized by intra-mural hematoma. This pathology was also observed in apoE+/+ x op male mice, and therefore, independent of hyper-lipidemia. No perceptible structural properties of aortas from op mice could be discerned prior to AngII infusion. Regional effects in the contractile response to phenylephrine were noted in aortic rings with enhanced responsivity in the upper thoracic aortas of op mice compared to those from +/+ mice. No differences in contractile response were noted in aortic rings from the lower thorax. In conclusion, deficiency of M-CSF attenuated AngII-induced atherosclerosis but led to an unanticipated pathology of intra-laminar hemorrhage in the upper aortic regions.